Gastrointestinal (GI) motility/functional disorders affect up to 25% of the US population. Common intestinal motility disorders include Irritable Bowel Syndrome and Fecal Incontinence, whereas more rare forms such as Hirschsprung's Disease have a genetic basis and are associated with absence or paucity of enteric nerves. Current treatment plans for GI motility/functional disorders range from changes in diet to bowel resection, however there are very few drugs available that target the primary deficiencies in controlled peristalsis. One barrier to research of GI disease is that it has largely relied on in vvo animal studies, which are intrinsically low throughput. Recently, we have established a culture system to generate human intestinal tissue organoids (HIOs) through directed differentiation of human embryonic and induced pluripotent stem cells (collectively called PSCs). HIOs are three-dimensional structures containing most epithelial and mesenchymal cell types found in the intestine. However, due to lack of an enteric nervous system in HIOs, the system is not a useful platform to study GI motility disorders. We hypothesize that the enteric nervous system can be built into HIOs by introducing neural crest stem cells (NCSC) into the differentiation process. There are several well-established methods to generate neural crest cells from PSCs in vitro and we propose to use PSC-derived NCSCs to construct human intestinal organoids containing enteric neurons and glial cells. In aim 1 we propose to generate human PSCs-derived vagal NCSC in vitro by modifying existing protocols that have been used to generate more anterior NCSCs. We will test the differentiation potential of PSC-derived NCSCs in vitro and following engraftment into chicken embryos. In aim 2, we will use NCSCs to generate human intestinal organoids containing enteric nerves. We will use several approaches to incorporate NCSCs into developing intestinal organoids by combining the two tissues during organoid development. We will also manipulate signaling pathways that function during embryonic ENS development to promote incorporation, proliferation and differentiation of NCSCs into ENS cell types in organoids. ENS formation will be analyzed by markers and by function. Development of an in vitro intestinal organ system containing an ENS would be an ideal platform for high throughput studies aimed at identifying new therapies to improve ENS function in patients with GI motility disorders. ! PUBLIC HEALTH RELEVANCE: Gastrointestinal (GI) motility/functional disorders affect up to 25% of the US population. Common intestinal motility disorders include Irritable Bowel Syndrome and Fecal Incontinence, whereas more rare forms such as Hirschsprung's Disease have a genetic basis and are associated with absence or paucity of enteric nerves. Current treatment plans for GI motility/functional disorders range from changes in diet to bowel resection, however there are very few drugs available that target the primary deficiencies in controlled peristalsis. One barrier to research of GI disease is that it has largely relied on in vvo animal studies, which are intrinsically low throughput. This proposal aims to generate human intestinal organoids containing functional epithelium, surrounded by smooth muscle that is innervated by enteric nerves. This research tool would allow for unprecedented studies of the cellular and molecular basis of gut motility in humans and be an ideal screening platform for drugs that regulate gut motility.